Sapogenin derivatives and preparation of same



Patented F eb. 19, 1946 SAPOGENIN DERIVATIVES AND PREPARATION OF SAME Russell Earl Marker, Mexico City, Mexico, and

Harry Means Crooks, Jr., Wittle, Detroit, Mich & Company, Detrol Michigan N Drawing.

and Eugene Leroy assignors to Parlie, Davis t, Mich., a corporation of Original application May 15, 1941, Serial No. 393,666. Divi May 24, 1944, Serial No 14 Claims. (of. 260-210) The invention relates to the preparation of glycosidic pseudo-sapogenln compounds. This application is a division of our copending application, Serial No. 393,666, filed May 15, 1941, now Patent No. 2,352,851, issued July 4, 1944.

In the copendlng application, Serial No. 393,667, filed May 15, 1941, of Russell Earl Marker, now Patent No. 2,352,852, issued July 4, 1944, it is shown that steroidal sapogenins may be isomerized, for example, by treatment with acetic anhydride for six to fifteen hours at about 200 C.,

to form a new class of sapogenin derivatives designated as pseudo-sapogenins.

According to the present invention, the pseudosapogenins or their ring A and/or B gly'cosidic derivatives are prepared by reacting glycosidic derivatives of the sapogenins with acidic agents, for example, acylating agents such as acid anhydrides, under conditions more vigorous than those required merely for acylation.

By glycosidic derivatives of the sapogenins we mean sapogenln derivatives in which sugar residues are attached through a hemi-acetal linkage to the cyclopentanoperhydrophenanthrene nucleus. In general, the exact nature of the structures of these substances are not known with certainty. The following formulae illustrate various types of the above sapogenin glycosides.

VIII. Sarsasaponln ded and this application 537,199

CH: CH: CII CH CH I' I A 0 (EH-CH:

l O--C a f H- CH $11011 ($11011 cnon o HOH JJHOH HOH 611* H- JJH, $111013 12:. Trillarln Generally speaking the glycosides of the steroidal sapogenins may be classified as (1) saponins;

( 2) simpler glycosides. The former usually con-' (Reinhold Publishing Corporation. New York city,

Since the steroidal sapogcnins occur in nature. not in the free form, but combined with sugar iii access system and in a position not known with greater certainty.

According to a further feature of our invention, the acylated glycosidic pseudo-sapogenin derivatives may be hydrolyzed with alkaline reagents with production of a glycosidic derivativeof a pseudo-sapogenin unacylated at least at the sac hydroml group and in the sugar residues.

Our invention may be more fully illustrated by the following examples.

Example 1 (a) The saponins otTrillium erectum or from Dioscorea oillosa may be isolated in the following manner.

l The powdered rhizomes are allowed to stand units as glycdsidic derivatives. the present inven tion makes it unnecessary to isolate the saposem ins. Instead, their more readily available glycosides may be converted directly to pseudo sapogenin derivatives. This elimination of a formerly essential step results in higher yields of steroidal hormones from plant sources.

The pseudo=sapogenins are characterized by the fact that they contain a new type or side chain which undergoes distinctive reactions. Thus the pseudo-sapogenins are unsaturated to bromine and therefore readily decolorize a solution of bromine in acetic acid. 0n treatment with acids, for example, with alcoholic hydrochloric acid, the pseudo-sapogenins are isomerized to the corresponding steroidal sapogenins. The side chain of the pseudo-sapogenins contains a reactive hydroxyl group which may be acylated, for example, acetylated.

It is believed that the properties of pseudosapogenins are best explained if the side chain attached to ring D of the cyclopentanoperhydro- 'phenanthrene nucleus be represented by one of thefollowins partial formulae: 7

om O--CHa oa -cnl carom cm o cm I tin-of on om Y UHe-QHB tom two days with sumcient alcohol to term a thin cream. This usually requires about 3 parts of alcohol per 1 part of powdered rhizome. After most of the alcohol-soluble material has been leached out in this manner, the cream is filtered and the filter cake washed well with alcohol. The alcohol solution is concentrated to a syrup and the syrup dissolved in about an equal weight or less oi. hot alcohol. After the solution has cooled, several volumes of ether or petroleum ether are added to cause precipitation of the crude glycoside and to dissolve the fat. The clear solution is decanted from the gummy precipitate and the latter then dissolved in alcohol and a warm alcoholic solution of a sterol such as chols esterol or sitosterol is added. After standing overnight most of the saponin has been precipitated in the form of a sterol adduct. This is collected and washed with a small amount of alcohol. After this addition compound has been air dried, it is dissolved in about 2 volumes of pyridine by warming on the steam bath for about fifteen minutes. After solution is complete about 5-10 volumes of other is added, causing precipitation of the free saponin. This is filtered and washed with ether. It may be purified by dissolving it inalcohol' or water and adding ether to repreclpltate the purified saponin. As thus obtained it is a white microcrystalline powder fairly soluble in alcohol and water but insoluble in ether and petroleum ether. With sterols having a 34s) =hydroxyl group it forms insoluble adducts.

One hundred and ty grams of the saponin from Trillium erectum is acetylated in the ioilowing fashion. The saponin is dissolved in 5 volumes of pyridine, and it volumes of acetic anhydride added. Alter heating on the steam bath for one hour, the mixture is poured into other and the ethereal solution washed with water to remove pyridineiand acetic acid. The ethereal layer is evaporated to leave a gummy residual acetate which after standing a few days and rubhing with a glass rod crystallizes. This product may be recrystallized from organic solvent and then has themelting-point of about 16 C.

(b) Cine hundred and fifty grams of the above acetylated saponin is dissolved in 100 cc. of acetic anhydride and heated in a bomb tube or auto clave at 200 C. for ten hours. Then the acetic anhydricle is removed under reduced pressure. This residue is the acetate of Trillium erecium pseudo-saponin.

(c) The above acetylated Trillium erectum pseudo-saponin is dissolved in 2 liters of glacial acetic acid and added at 30 C. to a stirred solution of 25 g. or" chromic anhydride in 1 liter of acetic acid. After the mixture has stood one and a halihours at this temperature, the excess chromic anhydride is destroyed by addition of zinc powder. The solution is filtered from excess zinc and the filtrate is concentrated in vacuo. The residuals dissolved in ether and washed with water and saturated sodium bicarbonate solution. The ethereal solution is evaporated to leave a clear yellow gummy residue weighing about 120 g. This residue consists essentially of the substance representable by the formula CH: CH CH2 I 6:0 l o on, g o A -d-cm-ont-on-om-o-o-om k/ g'iuooes glucose glucose (d) The above residue is dissolved in 1%; liters of 90% alcohol and a solution of 350 g. of potassium hydroxide in an equal amount of water is milligram. g i

of platinum black. in an atmosphere of nitrogen. Then the mixture is dissolved in boiling ligroin and set aside tocrystallize. The progesterone so obtained has a melting point of l27-8.5 C. and a biological activity of 1-2 International Units per Example 2 (a) a solution of 10 g. of diosgenin, 13 g. of bromacetylglucose and 5 g. of mercuric acetate in 120 cc. of dry benzene is refluxed for two hours.

Then the solution is evaporated to leave an oily residue and the latter is dissolved in ether. .The ethereal solution is concentrated to .a small volume and chilled. The crystalline precipitate which appears is collected and triturated with pression in melting added. The solution is refluxed'twenty minutes during which time a gum precipitates onthe sides of the flaslr. Then water is added to b the alcohol concentration down to about 60%. Then enough concentrated hydrochloric acid is added to neutralize the potassium hydroxide (litmus paper test) and to givean excess of about cc. of

. water is added and the Then the acetic anhydride is evaporated under reduced pressure and the residue is crystaliioed from methanol to yield 7-40 g. of o pregnadienol ii-dp)-one-20 acetate of melting point Hi -6 C.

(e) Seven grams of the above A -pregnadienol=3-(;3)-one-20 acetate is dissolved in about 500 cc. of alcohol and 7 g. oi a palladium barium sulfate catalyst containing 3% by weight of palladium is added. After shaking this mix= ture in a hydrogen atmosphere at about 30 his. pressure for half an hour, the hydrogenation is essentially complete. The solution is filtered and the residue concentrated and set aside to crystallize. lhus there is obtained A -pregnenol-3-(p) one-$0 acetate of melting point 149-431" C.

This may be hydrolyzed by refluxing it with 19 times its weight of 5% methanolic potassium hydroxide. The resultant solution is diluted with water and extracted with ether and the ether evaporated to leave the free hydroxy hetone. After recrystallization the n -pregnenol-t-(pb one-2ii thus obtained has a melting point of ltd-90 C. v

(f) Two grams of M-pregnenol-3- e one -im obtained for example as described above is heated for an hour at 200 d. with about half'its weight ether. The crude glucoside thus obtained is recrystallized Irommethanol and then. has a meltins, point of 197 C. It shows no depression'in melting point when mixedwith trillin acetate oi melting point 199-201 Trillium erectum. This glucoside is therefore diosgenin-a-dlucoside tetraacetate.

0n hydrolysis with 2% hydroxide the above glucoside tetraacetate yields trillin of melting point 250 C. and giving no depoint' when mixed with an authentic sample isolated from Trillium erectzim.

(b) A mixture of 5.2 g. oi trillin tetraalcetate and 15 cc. of acetic anhydride are heated in a bomb tube for ten hours at 200 C; Themixture then is distilled under reduced pressureto remove the acetic anhydride. The residue .may be crystallized from acetate oi melting point 165 C v (c) To a solution of 4 g..oi the pseudo-trillin acetate in 200 cc. of acetic acid cooled to 15 U.

is added with stirring a solution of 1.2. g. of chromic anhydrlde in 29100. of aceticacid. After the solution has stood for an honest 25 (2.,

product is extracted with ether.

water and 5% sodium hydroxide solution. Then the other is evaporated to leave a crystaliiueresi due. This is refluxed for nine y minutes'jwith, 50

cc. of alcohol containing 5 ccgoi concentrated. hydrochloric acid. Then the mixture is diluted with water, extracted with ether, the ethereal v layer washedwith water and sodium carbonate solution and then the ether removed on a steam bath. The residue is purified by treatment in the a known manner with Girards reagent and the hetone thus obtained distilled in a. high vacuum at -140" C- The distillate is crystallized from ether, acetone, and dilute methanol and thus 7 gives A -pregnadienol-3-(phone-30 of melting v 9} point lilo-212 Q.

This may be converted to ample, according to the method describedin the previous example. i

a sample 3 1. p (a) A mixture of it g. of sarsasapogenin, 13

a. of bromoa cetyl glucose and 5. e. of mercuric acetate in 120 cc. of dry benzene is refluxed for two hours. The solution is-evaporated in vacuo and the residue dissolved in ether. The ethereal solution is concentrated to a small volume and. chilled. The crystalline material which separates is collected, triturated with ether and I'ECI'YStBJ-r lized from alcohol togive sarsasapogenin-a-glucoside tetraacetate of M. P. 227 C 1 i The above tetraacetyl glucoside may-be hy drolyzed by letting it stand overnight with 2% methanolic potassium hydroxide solution. Thus C. as obtained i'roni' methanolic potassium The ethereal layer is washed well withv progesterone; for eX- there is obtained sarsasapogenin-a-glucoside or M. P. 245 C.

(b) Five grams of sarsasapogenin-a-glucoside tetraacetate and cc. of acetic anhydride are heated in a bomb tube for eight hours at 200 G. Then the acetic anhydride is removed in vacuo to leave a residue 01' pseudo-sarsasapogenin-aglucoside penta-acetate. This substance may be represented by the following structural formula,

thus formed is an acylated glycosidic pseudosapogenin derivative acylated at least at the exohydroxyl group and in the sugar residues.

This product may then be converted into other 5 useful products as set forth in our parent application, Serial No. 393,666 above referred to;

What we claim as our invention is:

l. The process which comprises isomerizing and acylating the side chain attached to ring D The above examples are intended to illustrate but not to limit the present invention and numerous variations in regard to starting materials, conditions of reaction, modes of isolation of the product and other details will be apparent to those skilled in the art after a perusal of this specification.

For example, as naturally occurring glycosidic derivatives of steroidal sapogenins which may be used in the practice of this invention there may be mentioned amolonin, sarsasaponin, digitonin, or like steroidal saponins Also, there may be used partially degraded glycosidic derivatives of these saponins, such as trillarin or trillin. Such partially degraded glycosidic derivatives of saponins are obtained .by hydrolyzing the saponin at some of the oligosacharide linkages by means of enzymes or dilute acids or similar reagents, Again, there may be used synthetic glycosidic derivatives of steroidal sapogenins such as the synthetic galactosides, glucosides, ribosides, and other glycosides oi sapogenins such as sarsasapogenin, diosgenin, or other steroidal sapogenins containing reactive nuclear hydroxyl groups. Synthetic glycosides suitable for the practice of this invention may also be prepared from sapdgenins which have reactive nuclear hydroxyl groups, but which are not aglycones of naturally occurring saponins. For example, although neither episarsasapogenins nor its glycosides occur in nature, glycosides of epi-sarsasapogenins may be prepared synthetically from sarsasaponin by converting the latter into its aglycone, sarsasapogenin, and then converting this into epi-sarsasapogenin. The epi-sarsasapogenin may then be treated to form the glycoside as for example by treatment with bromoac'etoglucose.

The conversion of the glycosidic derivative of thesteroidal sapogenin into an acylated glycosidic pseudo-sapogenin may be efl'ected by treating the former with an acylating agent under conditions more vigorous than those required for mere acylation. This step may be effected, for example, by treatment of the glycosidic derivatives of the sapogenins with a carboxylic anhydride at PIS-250 C. We have foundthat best results are obtained with lower fatty acid anhydrideswhile maintaining the reaction temperature in the neighborhood of 200 C. The product of a glycosidic derivative of a steroidal sapogenin by reacting said glycosidic derivative with a carboxylic acid anhydride at 175-250 C., with production of a glycosidic derivative of a pseudosapogenin acylated at least at the exo-hydroxyl group and in the sugar residue.

3. The process which comprises isomerizing and acylating the side chain attached to ring D of a glycosidic derivative of a-steroidal sapogenin by reacting said glycosidic derivative with a lower fatty acid anhydride at approximately 200 C., with production of a glycosidic derivative of a pseudo-sapogenin acylated at least at the exohydroxyl group andin the sugar residues.

4. The process which comprises subjecting a glycosidic derivative of a pseudo-sapogenin acylated at least at the exohydroxyl group and in the sugar residues-to hydrolysis with an alkaline reagent, production of a glycosidic derivative of a pseudo-sapogenin unacylated at least at the exo-hydroxyl group and in the sugar residues.

5. The process for the preparation of steroidal compounds which comprises isomerizing and acylating the side chain attached to ring D of so a glycosidic derivative of a steroidal sapogenin selected from the class consisting oi sarsasapogenin, and diosgenin, by reacting said glycosidic derivative with a carboxylic acid anhydride at 175-250 C., with production of a glycosidic derivative of a member of the class consisting of pseudo-sarsasapogenin, and pseudo-diosgenin,' said glycosidic pseudo-sapogenin derivative being 1 acylated at least at the exo-hydroxyl group and in the sugar residues.

compounds which comprises isomerizing' and acylating the side chain attached to ring D of a glycosidic derivative of a steroidal sapogenin selected from the class consisting of sarsasa- I pogenin, and diosgenin, by reacting said glyco- 6. The process for the preparation of steroidal sidic derivative with a lower fatty acid anhydride at approximately 200 C., with production of a glycosidic derivative of a member of the class consisting of pseudo-sarsasapogenin, and pseudodiosgenin, said glycosidic pseudo-sapogenin derivative being acylated at least at the exo-hydroxyl group and in the sugar residues.

7. The process of preparing an acylated Trillium erectum pseudo-saponin which comprises reacting a Trillium erectum saponin with an acylating agent under conditions more vigorousthan those required for mere acylation thereby obtaining a Trillium erectum pseudo-saponin acylated at the exo-hydroxyl group and in the sugar residues.

8. The process or preparing a 'pseudo-trillin acylate which comprises reacting a diosgenin glycoside with an acylating agent under conditions more vigorous than those required for mere acylation thereby obtaining a pseudo-trillin acylate.

9. The process of preparing an acylated pseudo-sarsasapogenin which comprises reacting a sarsasapogenin glucoside with an acylating agent under conditions more vigorous than those required for mere acylation thereby obtaining a pseudo-sarsasapogenin glucoside acylated at the exo-hydroxyl group and in the sugar residues.

10. A glycosidic pseudo-sapogenin acylated at least at the exo-hydroxyl group and in the sugar residues.

11. A glycosidic pseudo-sapogenin acetylated at least at the exo-hydronl group and in the sugar residues.

12. Completely acetylated Trillium erectum pseudo-saponin. v

13. Pseudo-sarsasapogenin a glucose pentaacetate.

14. Pseudo-trillin acetate. 7 RUSSELL EARL MARKER.

HENRY MEANS CROOKS. JR. EUGENE LEROY WIT'ILE. 

